1. Field of The Invention
The current invention concerns a novel type of gene therapy with small fragment homologous replacement. In particular, the invention concerns a method for introducing small fragments of exogenous DNA into regions of endogenous genomic DNA (cellular or an infecting pathogen) virtually homologous to the exogenous DNA. The exogenous DNA fragments contain sequence modifications that 1) correct mutations in the endogenous DNA or 2) introduce mutations that alter phenotype (cellular or of an infecting pathogen).
2. Background Art and Related Art Disclosures
Gene therapy as a means of treating human genetic disease has been brought into the forefront in recent years by its application for the treatment of a variety of diseases including adenosine deaminase deficiency (ADA) as described in Transplantation Proc., 23:170 (1991) and cystic fibrosis (CF) as described in Nature Med., 1:39 (1995), Nature Genet., 8:42 (1994), and Cell, 75:207 (1993).
Other human diseases which are potential targets for gene therapy include but are not limited to thalassaemias, sickle-cell anemia, xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and muscular dystrophy.
Cystic fibrosis (CF) is one of the diseases in which cDNA based gene therapy has been already proposed and studied. CF is an inherited disease afflicting about 1 in 2500 caucasian live births. The individuals afflicted with CF have a mean life expectancy of about 28 years. CF is characterized primarily by defective regulation of cAMP dependent chloride ion transport, most notably across the apical membranes of epithelial cells. CF results in a debilitating loss of respiratory and pancreatic function; the primary cause of death being an opportunistic Pseudomonas aeruginosa infection of the airways. The disease is associated with mutations in the cystic fibrosis transmembrane conductive regulator (CFTR) gene.
Isolation and characterization of the CFTR gene described in Science, 245:1059 and 1066 (1989) has been crucial for understanding the biochemical mechanism(s) underlying CF pathology. A mutation in exon 10 resulting in 3-bp, in-frame deletion eliminating a phenylalanine at codon 508 (.DELTA.F508) of CFTR protein, has been found in about 70% of all North American CF chromosomes (PNAS (USA), 87:8447 (1991).
Genetic complementation by gene replacement/conversion has been applied to alter the expression of endogenous mutant genes in cell lines. The CFTR cDNA has been transfected into cystic fibrosis cells to attempt complementation. While the CF phenotype was corrected with wild type CFTR cDNA by complementation, the transfection of the cDNA, however, does not actually correct the defective CFTR gene sequence. Introduction of an expression vector carrying the CFTR cDNA to attain complementation also does not ensure the effective regulation of the CFTR gene.
The use of a full-length wild type CFTR cDNA permits genetic complementation to occur and corrects the defective phenotype corresponding to mutations on CFTR (Cell, 68:143-155(1992)). The cAMP-dependent chloride ion transport defect associated with this and other CFTR gene mutations was corrected by introduction of wild type CFTR retrovirus-mediated cDNA into CF epithelial cells as described, for example, in Cell, 62:1227 (1990).
Correction of the defective Cl ion transport is the basis for all CF gene therapy strategies. CF gene therapy strategies have used adenovirus vectors for transfer into human nasal (Cell, 75:207 (1993) and lung airways (Nature Genet., 8:42 (1991)). A study using liposomes was also carried out in the nasal mucosa of CF patients Nature Med., 1:39 (1995). Each study demonstrated transient wtCFTR expression and correction of defective cAMP-dependent Cl transport.
Nature Medicine, 1:182 (1995); Nature Genet, 8:8 (1994) and Nature, 365:691 (1993) discuss pro and cons of cystic fibrosis gene therapy. More clinically oriented publications appear in Nature Genet. 9:126 (1995), Human Gene Therapy, 5:509 (1994), Human Gene Therapy, 5:1259 (1994); Gene Therapy, 2:29 (1995) and Gene Therapy, 2:38 (1995). These publications describe clinical trials and results of attempted gene therapy to correct cystic fibrosis using various delivery vehicles.
Numerous approaches have been proposed to facilitate gene therapy such as those described in Science, 244:1275 (1989), but at present all gene therapy clinical trials use cDNA-based DNA transfer strategies. Although cDNA-based gene therapy strategies have been shown to be relatively effective in the case of ADA, it is unclear whether a cDNA-based approach will be generally applicable for treatment of a broad spectrum of genetic disorders or whether it is even the optimal mode of gene therapy. Since the cDNA in the eukaryotic expression vector is under the regulation of a heterologous promoter/enhancer, it is expressed in a cell independent fashion and at levels that may not be appropriate for a given cell type.
Studies using recombinant viral vectors for gene therapy do not address concerns about host immune responses to prolonged or repeated exposure to the viral proteins. Liposome medicated gene therapy circumvents this issue, but like the viral approaches, it is cDNA-based, and does not assure cell-appropriate gene expression.
A potential drawback of cDNA approaches is expression and/or overexpression of gene's mRNA and production of protein in all cells with exogenous cDNA. There is evidence to suggest that under certain circumstances overexpression of a therapeutic cDNA can be toxic in vitro and in vivo (Pediatr. Pulmonol. Suppl., 9:30A (1993)). Eukaryotic expression vectors carrying cDNA are independent of cellular regulatory systems controlling RNA and protein levels and the resulting cell-independent expression of a therapeutic cDNA may activate metabolic and physiologic feedback mechanisms that are not compatible with normal function and/or survival. Since these questions remain unanswered, it is unclear whether the cDNA-based gene therapy strategies will ultimately result in a normal in vivo phenotype in treated individuals or be optimal for gene therapy.
It would, therefore, be advantageous to have available a method which could circumvent cell inappropriate expression and possible pathological consequences of such expression.
There have been previous attempts to alter the genome by homologous recombination between DNA sequences residing in the chromosome and newly introduced exogenous DNA sequences. Until now homologous recombination strategies have been primarily based on the transfer of homologous sequence into genomic DNA through replacement and insertion vectors and have relied on positive/negative selection protocols or on intrachromosomal recombination for generation of homologous recombination cells. This approach allowed the transfer of modifications of the cloned gene into the genome of a living cell (Science, 244:1288 (1989) and Int. J. Cell Clon., 8:80 (1990).
These existing, classical homologous recombination methods have not been efficient and this has been a major factor limiting the effectiveness of homologous recombination for gene therapy.
In the past, attempts to perform sequence-specific exchange of genomic DNA focussed on binding of oligonucleotides to desired sites in the DNA, and then on site specific cleavage of the genomic DNA. So far, only homopyrimidine or guanine-rich oligonucleotides have been successfully used with this technique.
Up to the present time, homologous recombination has therefore been generally limited in its application. Its primary application has been to the development of transgenic animals by introducing homologous recombination replacement and insertion vectors into mouse embryonic stem cells for gene inactivation and disruption experiments or for the correction of mutant genes in rodent cells (Cell, 56:313 (1990); PNAS, 86:8927 (1989); PNAS, 86:4574 (1989)).
This approach has not been successfully employed for gene correction in metabolically active human cells or in vivo given the disruption produced of intron sequences with selection markers. However, in vitro homologous recombination between two plasmids containing non-complementing, non-reverting deletions in an antibiotic resistance genes has been reported with extract from human cells (Mol. Cell Biol., 5:714 (1985)).
Single strand DNA (ssDNA) oligonucleotide fragments were also utilized to correct a mutation in nonintegrated plasmid DNA co-transfected into human cells (New Biologist, 1:223 (1989)). This approach was able to correct a single 14 nucleotide insertion mutation and the mutant phenotype with a 40 base single strand oligonucleotide. However, it does not achieve or demonstrate that this exchange can occur in chromatin DNA, in that these ssDNA fragments were not shown to cause a change in the cell phenotype when genes are integrated into or are a component of genomic DNA.
Single-stranded DNA coated with rec A was shown to anneal to homologous DNA and form a presumed triplex structure known as a "D-loop" in vitro (Science, 238:645 (1987)). The homologous pairing of genomic DNA sequences with a homologous rec A coated ssDNA fragment was inferred from the protection afforded the genomic HeLa cell DNA by the restriction enzyme Eco RI (Science, 254:1494 (1991)). The D-loop structure, however, was found to be unstable in vitro and may not have a long half-life in metabolically active cells.
Recently, in the U.S. Pat. No. 4,950,599 a method for exchanging homologous DNA sequences in a cell using DNA fragments encapsulated in polyoma capsid, a protein coat of a virus particle was described. While this approach uses DNA fragments, it does not recognize the necessity or importance of intron sequences within the exogenous fragment. The DNA fragments used in an in vitro study into hypoxanthine guanine phosphoribosyl transferase (HPRT) defective hematopoetic cells contained only exon sequences Mol. Cell Biochem. 92:107 (1990). The frequency of homologous recombination on this systems was only on the order of 10.sup.-5 and was not enhanced with recA coating or whether the DNA fragment was single or double stranded. In addition, it was unclear to what extent the exogenous DNA fragments were able to induce reversion to HPRT positive phenotype without spontaneous homologous recombination. Furthermore, the use of polyoma virus capsid for delivery of the exogenous DNA fragments will not circumvent issues related to host immune response.
In view of these inefficient attempts to correct mutated genes, it is clear that there is a need for a different in situ method of correcting genetic defects by homologous replacement in mammalian cells, particularly in human cells.
It would therefore be extremely useful to have available a method which would circumvent or eliminate all or some of the above listed disadvantages.
It is therefore a primary object of the current invention to provide a targeted gene replacement, using relatively small genomic DNA fragments with noncoding sequences flanking the sequences to be altered to replace endogenous sequences that are virtually homogenous. As implemented, the invention will circumvent cell inappropriate expression and the physiological and metabolic complications that result.
When applied in vivo, the present method is useful for gene therapy treating human genetic diseases and for countering the deleterious effects of these diseases. In addition, when applied in vitro the method is suitable for ex vivo gene therapy as well as for producing transgenic animals or the treatment of infectious diseases.